WO2003057729A2 - Mammal prolactin variants - Google Patents
Mammal prolactin variants Download PDFInfo
- Publication number
- WO2003057729A2 WO2003057729A2 PCT/EP2003/000448 EP0300448W WO03057729A2 WO 2003057729 A2 WO2003057729 A2 WO 2003057729A2 EP 0300448 W EP0300448 W EP 0300448W WO 03057729 A2 WO03057729 A2 WO 03057729A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hprl
- prolactin
- prl
- variant
- mutation
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2257—Prolactin
-
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57554—Prolactin
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- the invention relates to mammal prolactin (PRL) variants, and their use as antagonists of mammal prolactin receptors (PRLRs), more particularly of human prolactin receptor (hPRLR) .
- PRLRs mammal prolactin receptors
- hPRLR human prolactin receptor
- Prolactin is an anterior pituitary hormone involved in a wide spectrum of biological activities, among which are those related to lactation and reproduction (BOLE- FEYSOT et al . , Endocr. Rev., 19, 225-268, 1998).
- PRL actions on target tissues are mediated by a specific membrane-bound receptor, the Prolactin Receptor (PRLR) , which belongs to the cytokine receptor superfamily (KELLY et al . r Endocr. Rev., 12, 235-251, 1991).
- PRLR Prolactin Receptor
- PRL is also synthesized in extra-pituitary sites (for review, see BEN-JONATHAN et al . , Endocr. Rev., 17, 639-669, 1996) , such as mammary epithelial cells (GINSBURG and VONDERHAAR, Cancer Res., 55, 2591-2595, 1995) or prostate (NEVALAINEN et al . , J. Clin. Invest., 99, 618-627, 1997).
- extra-pituitary sites for review, see BEN-JONATHAN et al . , Endocr. Rev., 17, 639-669, 1996) , such as mammary epithelial cells (GINSBURG and VONDERHAAR, Cancer Res., 55, 2591-2595, 1995) or prostate (NEVALAINEN et al . , J. Clin. Invest., 99, 618-627, 1997).
- the hormone exerts a proliferative action on these cells (expressing the PRLR) via an autocrine/paracrine loop (GINSBURG and VONDERHAAR, Cancer Res., 55, 2591-2595, 1995; MERSHON et al . , Endocrinology, 136, 3619-3623, 1995; CLEVENGER and PLANK, J. Mammary Gland. Biol. Neopl., 2, 59-68, 1997).
- the growth-promoting activity exerted by PRL on some target tissues under normal conditions may be somehow involved in promoting tumor growth under pathological conditions.
- PRL Due to the failure of clinical treatments using dopamine agonists to reduce breast tumor progression (MANNI et al . , Breast Cancer Res. Treat., 14, 289-298, 1989), PRL has been considered for a long time as a minor player in human breast cancer.
- dopamine agonists fail to target extra-pituitary PRL synthesis, which now appears at least as important as circulating, pituitary-secreted PRL in these phenomena of tumor proliferation.
- PRLR antagonists able to compete with wild-type prolactin (WT-PRL) for receptor binding, but unable to trigger downstream signalling pathways, appears to be an alternative strategy to prevent, or at least reduce PRL-induced tumor proliferation, with potential implications in pathologies such as breast cancer and prostate hyperplasia (GOFFIN et al . , Mol. Cell. Endocrinol., 151, 79-87, 1999).
- WT-PRL wild-type prolactin
- GHR GH receptor
- binding sites 1 and 2 Two binding sites on the hormone, called binding sites 1 and 2, and proposed a model of PRLR activation by sequential homodimerization (GOFFIN et al . , Endocr. Rev., 17, 385-410, 1996).
- hPRLR human prolactin receptor
- G129R-hPRL This residual agonistic activity of G129R-hPRL was confirmed by the inventors in vitro using another proliferation assay (Ba/F3 cells transfected with the hPRLR encoding plasmid) , and in vivo in transgenic mice expressing G129R-hPRL analog: whereas PRLR-deficient mice are sterile and unable to develop a normal mammary gland (ORMANDY et al . , Genes De . , 11, 167-178, 1997), mice expressing G129R-hPRL analog fail to exhibit any reproductive deficiency and lactate successfully, clearly indicating that in vivo, G129R- hPRL does not abolish PRL-mediated actions.
- the inventors have undertaken to develop more efficient hPRLR antagonists. They have previously studied the potential involvement of the N-terminal tail of hPRL in its binding to the PRLR. They have engineered iterative N- terminal deletions in hPRL, ranging from removal of the 9 first residues up to the 14 first residues; the N-terminal sequences of wild type hPRL and hGH and of the deletion mutants of hPRL are shown on Figure 1.
- PRL SEQ ID No:l
- GH SEQ ID No: 2
- N-terminus is 9 residues longer in PRL, including a disulfide bond between Cys 4 and
- Cysii An arrow identifies putative helix 1 as predicted by homology modeling.
- deletion of the 9 first residues of hPRL slightly enhanced the affinity for the PRLR leading to increased maximal activity compared to wild-type hPRL (WT hPRL) in the luciferase assay, while deletion of the 14 first residues ( ⁇ l-14-hPRL) results in a decrease of the affinity and maximal activity (Endocrine Society 82 nd Annual Meeting, Toronto, June 21-24 2000, Abstract 613) .
- the inventors have now undertaken to test the effect of N-terminal deletions on the affinity and antagonistic activity of the G129R-hPRL analog. Therefore, they engineered two N-terminal deletions in G129R-hPRL, by removal of the 9 first residues (mutant ⁇ l-9-hPRL) and of the 14 first residues (mutant ⁇ l-14-hPRL) .
- the present invention provides an antagonist of a mammalian prolactin receptor, wherein said antagonist is a variant of mammal prolactin having the following mutations: a) a mutation or set of mutations within the 14 N-terminal amino acids, wherein said mutation or set of mutations prevents the formation of the disulfide bridge between Cys 4 and Cysn, and b) a sterically hindering mutation or set of mutations within binding site 2 of prolactin.
- Mutation (s) a) impairing the formation of the Cys 4 -Cysn disulfide bridge comprise for instance: deletions including Cys 4 and/or Cysu, or substitution of Cys 4 and/or Cysu by an amino acid other than a cystein.
- Mutation (s) b) comprise in particular any substitution of a small amino acid within binding site 2 of PRL by a large and/or charged amino acid in order to introduce a steric hindrance.
- mutations are for instance substitution of at least one residue among Glni22> Leui25, Se ⁇ 2 ⁇ r Ala 22 or Gly ⁇ 2 9, preferably Ala22/ more preferably Glyi29, by residues such as Tyr, Phe, Asp, Glu, Arg, Lys or Trp, preferably Arg, Lys or Trp.
- mutation (s) a) comprises the deletion of at least the 4 N-terminal residues, preferably of at least the 9 N- terminal residues of PRL.
- mutation (s) a) may further comprise the substitution of the Cysu residue by an amino acid other than a cystein. This further allows an easier purification of the variants, by avoiding aggregation thereof that may result from the presence of free SH groups.
- Preferred PRL variants of the invention are variants comprising the following mutations: a deletion of at least the 9 N-terminal residues and up to the 14 N-terminal residues; and a G129R substitution.
- the variants of the invention are variants of human prolactin (hPRL) .
- the present invention also provides polynucleotides encoding the PRL variants of the invention.
- Polynucleotides of the invention may be obtained by the well-known methods of recombinant DNA technology and/or of chemical DNA synthesis. These methods also allow to introduce the desired mutations in a naturally occurring DNA sequence.
- the invention also provides recombinant DNA constructs comprising a polynucleotide of the invention, such as expression cassettes wherein said polynucleotide is linked to appropriate control sequences allowing the regulation of its transcription and translation in a host cell, and recombinant vectors comprising a polynucleotide or an expression cassette of the invention.
- recombinant DNA constructs can be obtained and introduced in host cells by the well-known techniques of recombinant DNA and genetic engineering.
- the invention also comprises a prokaryotic or eukaryotic host cell transformed by a polynucleotide encoding a PRL variant of the invention.
- a PRL variant of the invention can be obtained by culturing a host cell containing an expression vector comprising a nucleic acid sequence encoding said PRL variant, under conditions suitable for the expression thereof, and recovering said variant from the host cell culture.
- the invention also provides transgenic non-human animals, in particular transgenic non-human mammals, transformed with a polynucleotide encoding a PRL variant of the invention.
- Suitable methods for the preparation of transgenic animals are for instance disclosed in: Manipula ting the Mouse Embryo, 2 nd Ed., by HOGAN et al . , Cold Spring Harbor Laboratory Press, 1994; Transgenic Animal Technology, edited by C. PINKERT, Academic Press Inc., 1994; Gene Targeting: A Practical Approach, edited by A.L. JOYNER, Oxford University Press, 1995; Stra tegies in Transgenic Animal Science, edited y G.M. MONASTERSKY and J.M. ROBL, ASM Press, 1995; Mouse Genetics : Concepts and Applica tions, by Lee M. SILVER, Oxford University Press, 1995.
- the invention also relates to a therapeutic composition
- a therapeutic composition comprising a PRL variant of the invention, or a polynucleotide encoding said PRL variant, optionally mixed with suitable carriers and/or excipient (s) .
- the PRL variants of the invention can further be conjugated to one or more chemical groups, in order to increase their molecular weight.
- suitable chemical groups include polyols, such as polyethylene glycol (PEG) or heterologous polypeptides preferably hydrosoluble polypeptides, such as serum albumin of fragments thereof.
- compositions of the invention are useful as PRLR antagonists, in particular for treating or preventing diseases involving PRLR-mediated effects, such as tumoral proliferation involving any form of benign or malignant tumor (hyperplasia, dysplasia, neoplasia, adenoma, carcinoma) in any PRL target tissue (breast, prostate, liver, pituitary, lymphocytes) , auto-immune diseases (lupus erythematosus, rheumatoid arthritis) , hyperprolactinemia, typically, any diseases arising from an oversti ulation of the PRLR (hypermastia, reproduction disorders) (BOLE-FEYSOT et al . , Endocr. Rev., 1998).
- diseases involving PRLR-mediated effects such as tumoral proliferation involving any form of benign or malignant tumor (hyperplasia, dysplasia, neoplasia, adenoma, carcinoma) in any PRL target tissue (breast, prostate
- compositions of the invention can be administered in various ways:
- a preferred route of administration is the parenteral route, including for instance intramuscular, subcutaneous, intravenous, intraperitoneal, or local intratumoral inj ections .
- the oral route can also be used, provided that the composition is in a form suitable for oral administration, able to protect the active principle from the gastric and intestinal enzymes.
- the therapeutic composition includes a polynucleotide encoding a PRL variant of the Invention, said nucleotide is generally inserted in an expression cassette allowing its expression in a target organ or tissue.
- the expression cassette can be directly transferred in the cells as naked DNA, or placed in an appropriate vector, such as a viral vector, for instance an adenovirus derived vector.
- Gene transfer can be performed ex vivo on cells removed from the subject to be treated and thereafter re- implanted into said subject, or can be performed by direct administration of the nucleic acid to said subject.
- the choice of the method of transfer and/or of the vector depends on the target organ or tissue, and/or on whether a short-time expression (transient expression) or a more stable expression is wanted.
- the amount administered will be chosen in order to supply a large excess of PRL variant over endogenous PRL in the blood and/or target tissue.
- high doses thereof can be administered, without risk of unwanted agonist effects. In most of cases, an amount of PRL variant resulting in a 10 to 100-fold excess over endogenous PRL will be suitable. If necessary, an amount of PRL variant resulting in a 1000-fold excess or more over endogenous can be administered.
- PRL wild-type and mutant forms
- WT hPRL the binding site 2 analog G129R-hPRL (Gly 129 replaced with Arg)
- G129R-hPRL Gly 129 replaced with Arg
- single N-terminal deleted mutants ⁇ l-9-hPRL, ⁇ l-10-hPRL, ⁇ l- 11-hPRL, ⁇ l-12-hPRL, ⁇ l-13-hPRL, ⁇ l-14-hPRL
- sequence of 5' primers are the following (5' to 3'): ⁇ l-9 (SEQ ID No:3): GGCATATGCGATCCCAGGTGACCCTTCG ⁇ l-10 (SEQ ID No:4) GGCATATGTCCCAGGTGACCCTTCGAG ⁇ 1-1KSEQ ID No:5) GGCATATGCAGGTGACCCTTCGAGACC ⁇ l-12 (SEQ ID No: 6) GGCATATGGTGACCCTTCGAGACCTGTT ⁇ 1-13(SEQ ID No:7) GGCATATGACCCTTCGAGACCTGTTTG
- the 3' primer is identical for all analogs; it corresponds to a sequence in the non-coding region of the hPRL cDNA, located in 3' of the unique HindiII restriction site(SEQ ID No:9): 5 'CTGTTACACCCACGCATGG 3' .
- PCR reaction was performed as follows: 200 ⁇ M dNTP; 45 ⁇ M MgCl 2 , 1.5 ⁇ l Taq Polymerase (5 u/ ⁇ l) , PCR buffer, 10 ng of template (plasmid pT7L-hPRL) , 20 pmoles of each primers. PCR was performed for 25 cycles: 94°C (30 sec), 56°C (30 sec), 72°C (1 min) . PCR products were subcloned into TA cloning vector (pCR II.1), then recombinant TA plasmids were digested using Ndel and HindiII and purified inserts were ligated into pT7L plasmid linearized using identical restriction enzymes. After transformation, E. coli BL21(DE3) colonies were analysed for their D ⁇ A content; plasmids were extracted and digested to confirm the presence of expected inserts, then sequenced to check the expected mutations. Production and purification of proteins
- Cell lysis was performed using a cell disintegrator (Basic Z, Cell D, Roquemaure, France) . Proteins were overexpressed as insoluble inclusion bodies that were solubilized in 8 M urea (5 min at 55°C, then 2 h at room temperature) and refolded by continuous dialysis (72 h, 4°C) against 50 mM NH 4 HC0 3 , pH 8.
- Protein purification was performed using chromatography equipment (GRADIFRAC) and columns (HITRAP Q SEPHAROSE, SEPHACRYL S200 High Resolution) purchased from AMERSHAM-PHARMACIA BIOTECH (Orsay, France) .
- the dialyzed proteins were centrifuged for at least 60 minutes (9000 x g) to remove aggregates before loading the cleared supernatant mixture onto an anion exchange HITRAP Q column (equilibrated in 50 mM NH 4 HC0 3 , pH 8 ) .
- PRLs eluted in two peaks, one major peak eluted at a concentration of 150 mM NaCl, and a minor one eluted at a higher salt concentration ( ⁇ 200 mM) .
- Analytical gel filtration of these fractions indicated that the major peak corresponds to monomeric PRL, whereas the minor one includes various multimeric forms.
- refolded (dialyzed) proteins were concentrated by tangential flow ultrafiltration using a YM10 MINIPLATE bioconcentrator (MILLIPORE CORP . -AMICON, Bedford, MA; 500 ml/min flow rate), then the concentrated solution was centrifuged (10 min, 9000 x g) to remove aggregates formed upon ultrafiltration.
- Supernatants were purified by gel filtration chromatography using a high resolution SEPHACRYL S-200 column equilibrated in 50 mM NHHC0 3 , 150 mM NaCl, pH 8. This second protocol usually led to lower yields due to higher protein precipitation upon the ultrafiltration step. Fractions corresponding to monomeric hPRLs (eluted from molecular sieve or anion exchange columns) were pooled, quantified, aliquoted and stored at -20°C.
- Protein size and purity were assessed using 15% SDS-PAGE under reducing (beta-mercaptoethanol) or non- reducing conditions. Protein fractions were quantified by Bradford protein assay (BIO-RAD Laboratories, Inc., Ivry-sur- Seine, France), using BSA as the reference.
- Double mutants Expression plasmids encoding analogs ⁇ 1-9-G129R- hPRL and ⁇ l-14-G129R-hPRL were constructed by substituting the EcoRI-BglII fragment from pT7L-G129R-hPRL plasmid (containing the G129R mutation) (GOFFIN et al . , J. Biol. Chem., 269, 32598-32606, 1994) for the corresponding EcoRI- Bgl l fragment in pT7L- ⁇ l-9-hPRL and pT7L- ⁇ l-14-hPRL expression vectors. Clones obtained were analysed for the presence of the insert, then sequenced to check the expected mutations.
- binding affinities were determined using cell homogenates of HL5 cells (expressing the human PRLR) , following the procedure previously described (KINET et al . , J. Biol. Chem., 274, 26033-26043, 1999).
- hPRL was iodinated using IODOGEN, and its specific activity was in the range of 40-50 ⁇ Ci/ ⁇ g. Binding assays were performed overnight at room temperature using 150-300 ⁇ g cell homogenate protein in the presence of 30,000 cpm [ 125 I]-hPRL and increasing concentrations of unlabeled competitor (WT or mutated hPRL) .
- IC 50 were 166 ⁇ 47 ng/ml for ⁇ l-9- G129R and 187 ⁇ 49 ng/ml for ⁇ 1-14-G129R, compared to 18 ⁇ 5 ng/ml (for WT hPRL) . None of the N-terminal deletion improves affinity compared to G129R-hPRL (single mutant) .
- the reference bioassay for lactogenic hormones is the lactogen-induced proliferation of rat Nb2 lymphoma cells.
- Rat Nb2 lymphoma cells were obtained from P. W. GOUT (Vancouver, Canada) and cultured as previously described
- Nb2 cells were routinely maintained in RPMI 1640 supplemented with 10% HS, 10% heat-inactivated FCS, 2 mM glutamine,
- the proliferation assay was performed as initially described (TANAKA et al . , J. Clin. Endocrinol .
- the assay was performed in 96-well plates using 2 x 10 4 cells/well on starting day, in a final volume of 200 ⁇ l, including hormones.
- Cell proliferation was estimated after 3 days of hormonal stimulation by adding 10 ⁇ l WST-1 tetrazolium salt (ROCHE, Meylan, France) .
- This survival reagent is metabolized by mitochondria of living cells, which leads to an increase in the OD measured at 450 nm (OD 450 ) in a manner that is proportional to the number of cells counted by hemocytometer (BERNICHTEIN et al . , Endocrinology, 142, 3950- 3963, 2001) .
- the experiments were performed at least three times in triplicate or quadruplicate.
- Human PRLR trans ⁇ riptional bioassay (HL5) Clone HL5 are 293 HEK fibroblasts stably transfected with plasmids encoding the human PRLR and a PRL- responsive reporter gene (containing the sequence encoding the luciferase gene under the control of a six-repeat sequence of the lactogenic hormone response element (LHRE) which is the DNA-binding element of STAT5 (KINET et al . , J. Biol. Chem., 274, 26033-26043, 1999).
- LHRE lactogenic hormone response element
- the HL5 clone was routinely cultured in DMEM-Nut F12 medium supplemented with 10% FCS, 2 mM glutamine, 50 U/ml penicillin, 50 ⁇ g/ l streptomycin, and 700 ⁇ g/ml G-418 (clonal selection) .
- the assay was performed in 96-well plates using 5 x 10 4 cells/100 ⁇ l/well in medium containing only 0.5% FCS. Cells were allowed to adhere overnight, then 100 ⁇ l hormones diluted in FCS-free medium were added to each well.
- Ba/F3 cells are mouse pro-B lympho ⁇ d cells dependent on Interleukin-3 (IL-3) for growth.
- Ba/F3-hPRLR cells were obtained after transfection using a plasmid encoding the hPRLR, and a double selection involving G-418 treatment and substitution of hPRL for IL-3 in the growth medium. Cells were transfected (electroporation) using the plasmid encoding the hPRLR and the population stably expressing the receptor was selected after G-418 treatment.
- Ba/F3-hPRLR cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 50 U/ml penicillin, 50 ⁇ g/ml streptomycin, 500-1000 ⁇ g/ml G- 418, and 10 ng/ml WT-hPRL instead of IL-3.
- Optimal conditions of bioassay were determined using WT hPRL as ligand, and are the following: before the proliferation assay, cells were starved for 6 hours in 1% FCS RPMI medium (with additives) , then distributed in 96 well-plates at a density of 5 x 10 4 cells/well in a final volume of 100 ⁇ l in the same medium (excluding hormones) .
- agonism experiments 100 ⁇ l of [2x] hormones were added; in antagonism experiments, 50 ⁇ l of [4x] hormones to be tested for antagonistic properties and 50 ⁇ l of [4x] WT hPRL (final concentration of 10 ng/ml) were added.
- Cell proliferation was monitored after 3 days of hormonal stimulation using 10 ⁇ l of WST-1. Experiments were performed at least three times in triplicate or quadruplicate. Results
- monomeric hPRL induces cell proliferation in the classical Nb2 cell proliferation assay with a maximal effect at 1-2 ng/ml.
- Figure 3A shows cell proliferation in presence of increasing concentrations of hPRL (—•—) , ⁇ l-9-hPRL (—D—) and ⁇ l-14-hPRL (— ⁇ —) ;
- Figure 3B shows cell proliferation in presence of increasing concentrations of hPRL (—• ⁇ —) , ⁇ l-10- hPRL ( —*— ), ⁇ l-11-hPRL (—O—) , ⁇ l-12-hPRL (--D--) , ⁇ l- 13-hPRL (—O—) .
- Ba/F3-hPRLR cell prolifera tion bioassay In contrast to the assay with Nb2 cells, the hPRLR-mediated proliferation assay with Ba/F3 cells displayed different mitogenic activities of the analogs. WT hPRL induced growth of this cell population in a dose-dependent manner, with maximal effect at ⁇ 10 ng/ml, which correlates with the cell selection by substituting 10 ng/ml hPRL for IL- 3 in routine culture medium.
- Figure 4A shows proliferation of Ba/F3 cells in presence of increasing concentrations of hPRL (—•—) , ⁇ l-9- hPRL (—D—) and ⁇ l-14-hPRL (— ⁇ —) ;
- Figure 4B shows proliferation of Ba/F3 cells in presence of increasing concentrations of hPRL (—•—), ⁇ l-10-hPRL ( —*— ), ⁇ l-11- hPRL (—O—) , ⁇ l-12-hPRL (—D—) , ⁇ l-13-hPRL (—O—) .
- FIG. 5 depicts hPRL transcriptional activity (% of activity vs . WT hPRL maximal effect referred as 100%) in presence of increasing concentrations ( ⁇ g/ml) of: hPRL (—•—), ⁇ l-9-hPRL _ (-D-) , ⁇ l-14-hPRL (- ⁇ -) , ⁇ l-10-hPRL ( —* — ), ⁇ l-11-hPRL (—O—), ⁇ l-12-hPRL (—D— ) , ⁇ l-13-hPRL (—O— ) .
- results are expressed in fold induction of luciferase activity (i.e. percentage of activity vs WT hPRL maximal effect referred as 100%) .
- ⁇ l-14-hPRL was less active than hPRL, regarding both its dose-response curve (EC 5 o ⁇ 3-fold higher) and its maximal activity (60 % of WT hormone) .
- Nb2 cell proliferation assay AGONISM Nb2 cell proliferation assay AGONISM.
- Figure 6 shows cell proliferation without hPRL (D) and in presence of increasing concentrations of purified WT hPRL ( ⁇ ) , G129R-hPRL ( ⁇ ) , ⁇ l-9-G129R-hPRL ( _ ⁇ ) and ⁇ l-14-G129R-hPRL (ES)
- WT hPRL induces maximal proliferation at 1- 2 ng/ml, whereas the dose-dependent mitogenic effect of G129R-hPRL is shifted to the high concentrations, but reaches (sub) maximal proliferation.
- both double N-Terminal deleted mutants ⁇ l-9-G129R-hPRL and ⁇ 1-14-G129R- hPRL are devoid of significant agonistic activity.
- the agonistic dose- response curve obtained with G129R-hPRL is shifted by more than two log units to the right compared to WT hPRL, with maximal effect achieved at about 0,5 to 1 ⁇ g/ml.
- Figure 7A shows activation of the LHRE- luciferase reporter gene by increasing concentrations of WT hPRL ( ⁇ ), and the three Gl29R-containing analogs, G129R-hPRL G129R-hPRL ( ⁇ ), ⁇ l-9-G129R-hPRL (H ) , and ⁇ l-14-G129R-hPRL (S3).
- G129R-hPRL The agonistic activity of G129R-hPRL is extremely reduced in this assay, reaching a maximal level ⁇ 2% of hPRL activity. Similarly, none of the double mutant induced detectable level of luciferase activity, even when tested at extremely high concentrations (up to 50 ⁇ g/ml) .
- Figure 8A shows cell proliferation in presence of increasing concentrations of purified WT hPRL ( ⁇ ), G129R-hPRL ( ⁇ ) , ⁇ l-9-G129R-hPRL ( _ ⁇ ) , and ⁇ 1-14-G129R- hPRL ( ⁇ ) .
- G129R-hPRL induced sub-maximal proliferation with a dose-response curve displaced by 2 logs to the high concentrations.
- none of the double mutants ⁇ l- 9-G129R-hPRL and ⁇ l-14-G129R-hPRL induced significant proliferation.
- the curve obtained for G129R-hPRL was displaced to the right by ⁇ 2 log units and achieved sub-maximal (50-80%) level compared to hPRL.
- hPRL and G129R-hPRL displayed bell-shaped curves, a typical observation when using these ligands (KINET et al . , Recent Res. Devel. Endocrinol . , 2, 1-24, 2001).
- Both ⁇ l-9-G129R-hPRL and ⁇ l-14-G129R-hPRL failed to display any agonistic activity, even at concentration as high as 10 ⁇ g/ml.
- Antagonistic assays were performed by competing a fixed concentration of WT hPRL (10 ng/ml) with increasing concentrations of the analogs.
- Figure 8B shows cell proliferation in presence of increasing concentrations of ⁇ l-9-G129R-hPRL (- ⁇ -), ⁇ l-14-G129R-hPRL (-A-) , G129R- hPRL (— ⁇ —) competing with the fixed concentration of WT hPRL.
- G129R-hPRL displays a significant agonistic activity
- the inhibitory effect observed in competition assays presumably reflects a combination of real antagonism and self-antagonism phenomenon (GOFFIN et al . , J. Biol. Chem., 269, 32598-32606, 1994; BERNICHTEIN et al . , Endocrinology, 142, 3950-3963, 2001, KINET et al . , Recent Res. Devel. Endocrinol., 2, 1-24, 2001).
- EXAMPLE 4 ⁇ 1-9-G129R INHIBITS PRL-INDUCED MAPK ACTIVATION IN LIVER FROM WILD TYPE BALB-C/J MOUSE
- Membranes were blocked with 5% skimmed milk for lh at room temperature, and after extensive washing, they were incubated overnight with a primary monoclonal antibody specifically directed against the active forms of Erkl and Erk2 MAP kinases (phosphorylated on threonine 202 and tyrosine 204 ) . After extensive washing, membranes were incubated for 1 h with secondary horseradish peroxidase
- HRP horseradish peroxidase
- ECL enhanced chemiluminescence
- Membranes were then dehybridized using stripping buffer for 30 min at 50 °C, and after washing and reblocking, they were reprobed using the polyclonal anti-Erkl/Erk2 antibody which recognizes both active and inactive forms of Erkl and Erk2 MAP kinases. Then, the blots were revealed after incubation with an HRP-conjugated anti-rabbit antibody.
- Top panel (P-Stat5 and P-Stat3) : phosphorylated Stat5 and phosphorylated Stat3;
- This in vivo bioassay uses transgenic mice (males) expressing human PRL or G129R-hPRL under the control of the ubiquitous metallothionein promoter (Tg Met-hPRL and
- Tg Met-G129R Tg Met-G129R
- Tg Prob-rPRL prostate-specific probasin promoter
- MAPK-P phosphorylated MAPK
- MAPK total MAPK
- Figure 11 shows that Gl29R-hPRL displays an agonistic activity in vivo comparable to that of wild type prolactin.
- Antagonist ⁇ l-9G129R-hPRL
- MAPK-P phosphorylated MAPK in the prostate ventral and dorsolateral lobes and in the presence (+) or absence (-) of ⁇ l-9G129R-hPRL mutant;
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- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Rheumatology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Pain & Pain Management (AREA)
- Physical Education & Sports Medicine (AREA)
- Reproductive Health (AREA)
- Diabetes (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE60326430T DE60326430D1 (en) | 2002-01-08 | 2003-01-08 | VARIANTS OF MAMMAL'S PULACTIN |
CA2472647A CA2472647C (en) | 2002-01-08 | 2003-01-08 | Mammal prolactin variants |
EP03729258A EP1463758B1 (en) | 2002-01-08 | 2003-01-08 | Mammalian prolactin variants |
JP2003558043A JP4324477B2 (en) | 2002-01-08 | 2003-01-08 | A variant of mammalian prolactin |
US10/500,968 US7785838B2 (en) | 2002-01-08 | 2003-01-08 | Variants of mammalian prolactin, prolactin receptor antagonists, and methods of use |
AU2003215539A AU2003215539B2 (en) | 2002-01-08 | 2003-01-08 | Mammal prolactin variants |
MXPA04006676A MXPA04006676A (en) | 2002-01-08 | 2003-01-08 | Mammal prolactin variants. |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02290030.2 | 2002-01-08 | ||
EP02290030A EP1325930A1 (en) | 2002-01-08 | 2002-01-08 | Mammal prolactin variants |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2003057729A2 true WO2003057729A2 (en) | 2003-07-17 |
WO2003057729A3 WO2003057729A3 (en) | 2003-10-16 |
Family
ID=8185691
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2003/000448 WO2003057729A2 (en) | 2002-01-08 | 2003-01-08 | Mammal prolactin variants |
Country Status (11)
Country | Link |
---|---|
US (1) | US7785838B2 (en) |
EP (2) | EP1325930A1 (en) |
JP (1) | JP4324477B2 (en) |
CN (1) | CN100358919C (en) |
AT (1) | ATE424411T1 (en) |
AU (1) | AU2003215539B2 (en) |
CA (1) | CA2472647C (en) |
DE (1) | DE60326430D1 (en) |
ES (1) | ES2323233T3 (en) |
MX (1) | MXPA04006676A (en) |
WO (1) | WO2003057729A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008114077A1 (en) * | 2007-03-20 | 2008-09-25 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Constitutively active mutants of the prolactin receptor |
WO2011151405A1 (en) | 2010-06-04 | 2011-12-08 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Constitutively active prolactin receptor variants as prognostic markers and therapeutic targets to prevent progression of hormone-dependent cancers towards hormone-independence |
US11477969B2 (en) | 2013-08-27 | 2022-10-25 | Recombinetics, Inc. | Efficient non-meiotic allele introgression in livestock |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5888832B2 (en) * | 2005-12-22 | 2016-03-22 | フラームス・インテルウニフェルシタイル・インステイチュート・フォール・ビオテヒノロヒー・ヴェーゼットウェー(ヴェーイーベー・ヴェーゼットウェー)Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw(Vib Vzw) | Means and methods for mediating protein interference |
AU2006326940B2 (en) * | 2005-12-22 | 2012-04-19 | Vib Vzw | Means and methods for mediating protein interference |
EP2332995A1 (en) * | 2009-12-10 | 2011-06-15 | Bayer Schering Pharma Aktiengesellschaft | Neutralizing prolactin receptor antibodies and their therapeutic use |
US10920242B2 (en) | 2011-02-25 | 2021-02-16 | Recombinetics, Inc. | Non-meiotic allele introgression |
EP2530089A1 (en) * | 2011-06-03 | 2012-12-05 | Bayer Pharma Aktiengesellschaft | Neutralising prolactin receptor antibody Mat3 and its therapeutical use |
EP2846822A2 (en) * | 2012-05-11 | 2015-03-18 | Prorec Bio AB | Method for diagnosis and treatment of prolactin associated disorders |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4767709A (en) * | 1984-06-28 | 1988-08-30 | The Johns Hopkins University | Growth-related hormones |
-
2002
- 2002-01-08 EP EP02290030A patent/EP1325930A1/en not_active Withdrawn
-
2003
- 2003-01-08 EP EP03729258A patent/EP1463758B1/en not_active Expired - Lifetime
- 2003-01-08 CN CNB038033577A patent/CN100358919C/en not_active Expired - Fee Related
- 2003-01-08 US US10/500,968 patent/US7785838B2/en not_active Expired - Fee Related
- 2003-01-08 DE DE60326430T patent/DE60326430D1/en not_active Expired - Lifetime
- 2003-01-08 AU AU2003215539A patent/AU2003215539B2/en not_active Ceased
- 2003-01-08 AT AT03729258T patent/ATE424411T1/en not_active IP Right Cessation
- 2003-01-08 CA CA2472647A patent/CA2472647C/en not_active Expired - Fee Related
- 2003-01-08 ES ES03729258T patent/ES2323233T3/en not_active Expired - Lifetime
- 2003-01-08 JP JP2003558043A patent/JP4324477B2/en not_active Expired - Fee Related
- 2003-01-08 MX MXPA04006676A patent/MXPA04006676A/en active IP Right Grant
- 2003-01-08 WO PCT/EP2003/000448 patent/WO2003057729A2/en active Application Filing
Non-Patent Citations (4)
Title |
---|
A. TCHELET ET AL.: "Selective Modification at the N-terminal Region of Human Growth Hormone That Shows Antagonistic Activity" MOLECULAR AND CELLULAR ENDOCRINOLOGY, vol. 130, 1997, pages 141-152, XP002198975 * |
M. LLOVERA ET AL.: "Involvement of prolactin in breast cancer: redefining the molecular targets" EXPERIMENTAL GERONTOLOGY, vol. 35, 2000, pages 41-51, XP002198974 * |
S. BERNICHTEIN ET AL.: "N-Terminal Deletion in Human Prolactin Enhances Biological Activity and Reduces Formation of Covalent Multimers" ENDO 2000, ENDOCRINE SOCIETY 82ND ANNUAL MEETING, TORONTO, June 2000 (2000-06), page 153 XP001070977 cited in the application * |
V. GOFFIN ET AL.: "Antagonistic Properties of Human Prolactin Analogs That Show Paradoxical Agonistic Activity in the Nb2 Bioassay" J. BIOL. CHEM., vol. 271, no. 28, 12 July 1996 (1996-07-12), pages 16573-16579, XP002921797 cited in the application * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008114077A1 (en) * | 2007-03-20 | 2008-09-25 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Constitutively active mutants of the prolactin receptor |
US8263340B2 (en) | 2007-03-20 | 2012-09-11 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Constitutively active mutants of the prolactin receptor |
WO2011151405A1 (en) | 2010-06-04 | 2011-12-08 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Constitutively active prolactin receptor variants as prognostic markers and therapeutic targets to prevent progression of hormone-dependent cancers towards hormone-independence |
US11477969B2 (en) | 2013-08-27 | 2022-10-25 | Recombinetics, Inc. | Efficient non-meiotic allele introgression in livestock |
Also Published As
Publication number | Publication date |
---|---|
CN100358919C (en) | 2008-01-02 |
MXPA04006676A (en) | 2005-03-31 |
WO2003057729A3 (en) | 2003-10-16 |
DE60326430D1 (en) | 2009-04-16 |
CA2472647A1 (en) | 2003-07-17 |
JP2005526493A (en) | 2005-09-08 |
ES2323233T3 (en) | 2009-07-09 |
JP4324477B2 (en) | 2009-09-02 |
US7785838B2 (en) | 2010-08-31 |
AU2003215539B2 (en) | 2009-03-26 |
CA2472647C (en) | 2013-04-02 |
AU2003215539A1 (en) | 2003-07-24 |
US20060277614A1 (en) | 2006-12-07 |
EP1463758A2 (en) | 2004-10-06 |
ATE424411T1 (en) | 2009-03-15 |
EP1325930A1 (en) | 2003-07-09 |
EP1463758B1 (en) | 2009-03-04 |
CN1628128A (en) | 2005-06-15 |
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